Hirschsprung's disease (HD), congenital absence of neurons in the distal intestinal tract, is a potentially lethal birth defect that afflicts 1/5000 liveborn humans. HD is thought to be a consequence of failed colonization of the hindgut by ganglion cell precursors (neuroblasts) derived from the neural crest. The processes which regulate the migration, proliferation, and differentiation of enteric neuroblasts are not understood, but probably involve dynamic interactions between these cells and the surrounding intestinal mesenchyme. Lethal spotted (ls), piebald lethal (sl), and dominant megacolon (Dom) are three alleles associated with HD in mice. I hope to learn more about the pathogenesis of HD through study of these murine models. In the proposed studies, enteric neuroblasts, their microenvironment, and interactions between the two will be examined by using expression of the transgene, D/beta/H- nlacZ, as a marker of neuroblasts in wild-type mice and ls/ls, sl/sl, and Dom/+ embryos. Use of the transgene is a novel approach which permits positive identification of "undifferentiated" enteric neuroblasts in intact gut and histological sections during the critical stages of embryogenesis when the gut is colonized. A combination of immunohistochemical, ultrastructural, autoradiographic techniques will be applied to define morphological and biochemical features which distinguish enteric neuroblasts and to identify possible differences in these cells or surrounding mesenchyme between wild-type, ls/ls, sl/sl, and Dom/+ embryos. The nature of the defect in sl/sl and Dom/+ embryos will be examined by examining chimeric mice produced by aggregating transgenic mutant embryos with non-transgenic wild-type embryos. We recently published such studies of ls/ls-wild type chimeras which demonstrated that the primary defect caused by the ls mutation is not cell autonomous (intrinsic to migrating neuroblasts), but is environmental (extrinsic anomalies in the milieu through which neuroblasts migrate). The effects of environmental manipulations on neuroblast colonization in wild-type and mutant embryonic intestines engrafted under the renal capsule will also be investigated with the intent of finding growth conditions that "rescue" the mutant phenotype. The cumulative results of these studies will provide insight into the pathogenesis of HD and direct future studies to identify specific genetic or environmental factors responsible for HD in humans.